The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid. Since its isolation more than 100 years ago, Streptococcus pneumoniae has been one of the more intensively studied microbes. For example, much of our early understanding that DNA is, in fact, the genetic material was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe. Despite the vast amount of research with S. pneumoniae, many questions concerning the virulence of this microbe remain. It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics.
The frequency of Streptococcus pneumoniae infections has risen dramatically in the past 20 years. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Streptococcus pneumoniae strains which are resistant to some or all of the standard antibiotics. This has created a demand for both new anti-microbial agents and diagnostic tests for this organism.
While certain Streptococcal factors associated with pathogenicity have been identified, e.g., capsule polysaccharides, peptidoglycans, pneumolysins, PspA Complement factor H binding component, autolysin, neuraminidase, peptide permeases, hydrogen peroxide, IgA1 protease, the list is certainly not complete. Moreover, very little is known concerning the temporal expression of such genes during infection and disease progression in a mammalian host. Discovering the sets of genes the bacterium is likely to be expressing at the different stages of infection, particularly when an infection is established, provides critical information for the screening and characterization of novel antibacterials which can interrupt pathogenesis. In addition to providing a fuller understanding of known proteins, such an approach will identify previously unrecognised targets.
The hydrolysis of peptide bonds is a central metabolic activity in bacterial cells. E. coli contains at least 40 enzymes that catalyze this reaction with representatives in every cellular compartment (Maurizi, M. R. (1992) Experientia, 48, 178-201; Gottesman, S. and Maurizi, M. R. (1992) Microbiol. Rev., 56, 592-621). These enzymes participate in a diverse array of processes. In growing cells, some substrates are degraded to rid the cell of damaged or nonfunctional proteins or peptides, whereas in other cases degradation is used to regulate the level of functional proteins. In starving cells, functional (although perhaps not functioning) proteins that are not normally degraded become susceptible to degradation. In both circumstances, proteases and peptidases play an indispensable role on the survival of the cell and therefore, inhibitors of these activities would prevent the bacterium from establishing and maintaining infection of the host and thereby have utility in anti-bacterial therapy.
Clearly, there is a need for factors, such as the novel compounds of the invention, that have a present benefit of being useful to screen compounds for antibiotic activity. Such factors are also useful to detemiene their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases.
The polypeptides of the invention have amino acid sequence homology to a known B. subtilis aminopeptidase protein. See Winters et al., "The ampS-nprE (124-127 degrees) region of the Bacillus subtilis 168 chromosome: Sequencing of a 27 kb segment and identification of several genes in the area", Microbiology 142, 3033-3037 (1996); Genbank, Accession U51911. Also see Motoshima et al., "Molecular cloning and nucleotide sequence of the aminopeptidase T gene of Thermus aquaticus YT-1 and its high-level expression in Escherichia coli", Agric. Biol. Chem., 54(9):2385-2392 (1990).